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TotalSeq™-B or -C Cell Surface and Nuclear Protein Staining with 10x Chromium Single Cell Flex Gene Expression

 

The following protocol describes combined cell surface and nuclear protein staining with TotalSeq-B or -C antibodies, with or without TotalSeq hashtag antibodies, using 10x Genomics Chromium Single Cell Flex Gene Expression assay. This protocol has been optimized using liquid, single-antibody TotalSeq-B or -C conjugates, and lyophilized TotalSeq-B or -C cocktails.

 

DO NOT combine TotalSeq-B and TotalSeq-C antibodies in a single experiment. This protocol has not been optimized for the staining of proteins encapsulated in extracellular vesicles like cytokines and chemokines or phosphorylated proteins, however it is compatible with some cytoplasmic proteins. Contact BioLegend Technical Services for guidance concerning compatible cytoplasmic proteins.

 

Critical:

  • It is critical to have as many cells as possible when starting the nuclear staining portion of this protocol, so we advise starting the cell surface antibody staining with 2 x 106 cells/sample. Alternatively, you can also combine uniquely hashtagged samples following cell surface protein staining to start the nuclear antibody staining step with 2 x 106 cells.
  • After completing cell surface protein staining, a 1-hour fixation step is required before staining nuclear proteins.
  • Following fixation, you may either proceed immediately with nuclear protein staining or store the samples overnight and continue staining the next day.
  • If you intend to perform both surface and nuclear protein staining on the same day, ensure that you prepare the nuclear staining buffers and nuclear antibody pool before starting with the fixation step as described.
  • If you want to stop for the day after fixation prepare only the TotalSeq Diluent Solution and Sample Storage Buffer. Prepare remaining solutions the following day before starting the protocol. Please plan accordingly.

 

Read through this protocol and the associated 10x Genomics user guides in their entirety. The 10x Genomics Fixed RNA Profiling Reagent Kit user guide required is dependent on the TotalSeq format you will be using.

 

10x Genomics Fixation user guide required for any TotalSeq format:

10x Genomics kit user guide compatible with TotalSeq-B:

10x Genomics kit user guides compatible with TotalSeq-C:

 

Note:

TotalSeq-B antibodies are not intended for use with 10x Chromium Flex Barcode Oligo Capture or any GEM-X user guides. TotalSeq-C antibodies are not intended for use with the 10x Chromium Flex Feature Barcode technology for protein protocol.

 

Disclaimers

  • 10x Genomics has not validated this protocol and will provide limited support to customers using this protocol.
  • 10x Genomics has not validated the use of TotalSeq Hashtags with Flex gene Expression and will only provide limited customer support on use of hashtags. If you plan to use TotalSeq-C hashtags in combination with 10x Antibody Multiplexing barcodes for sample multiplexing, please note that BioLegend has not validated this method and can only offer limited guidance on demultiplexing the data.

 

Protocol Overview


Image showing TotalSeq Nuclear Staining workflow diagram

 

 

Reagent and Consumable List

  • Cell Staining Buffer (BioLegend, Cat. No. 420201)
  • Human TruStain FcX™ (Fc Receptor Blocking Solution) (BioLegend, Cat. No. 422301)
  • TruStain FcX™ PLUS (anti-mouse CD16/32) (BioLegend, Cat. No. 156603)
  • Custom TotalSeq Nuclear Buffer Set (BioLegend, Custom part No. 900012746). To order, contact BioLegend Custom Solutions.
    • TotalSeq Cell Resuspension Buffer
    • TotalSeq Fixation Buffer
    • TotalSeq Diluent
    • TotalSeq Nuclear Perm/Wash Component A
    • TotalSeq Nuclear Perm/Wash Component B
    • True-Stain Monocyte Blocker™
  • Cyto-Last™ Buffer (BioLegend, Cat. No. 422501)
  • UltraPure™ Salmon Sperm DNA Solution (ThermoFisher Scientific, Cat. No. 15632011 or equivalent)
  • UltraPure™ DNase/RNase-Free Distilled Water (ThermoFisher Scientific, Cat. No. 10977015 or equivalent)
  • Vanadyl ribonucleoside complexes solution (VRC) (NewEngland BioLabs, Cat. No. S1402S or equivalent)
  • Poly (vinylsulfonic acid, sodium salt) solution (PVSA) (MilliporeSigma, Cat. No. 278424-250ML or equivalent)
  • 1.5 mL Low Protein Binding Microcentrifuge Tubes (ThermoFisher Scientific, Cat. No. 90410 or equivalent)
  • 12 x 75 mm Falcon™ Round-Bottom Polystyrene Tubes with cap (Fisher Scientific, Cat. No. 14-959-1A or equivalent)
  • RNaseZap (ThermoFisher Scientific, Cat. No. AM9780 or equivalent)

 

Additional Suggested Reagents

  • TotalSeq-B Human Universal Cocktail, V2.0, (BioLegend, Cat No. 399917)
  • TotalSeq-B Human Universal Cocktail, V1.0, (BioLegend, Cat No. 399904)
  • TotalSeq-B Human Essential Cocktail, V1.0, (BioLegend, Cat No. 399918)
  • TotalSeq-B Human TBNK Cocktail, (BioLegend, Cat. No. 399902)
  • TotalSeq-B Mouse Universal Cocktail, V1.0, (BioLegend, Cat. No. 199902)
  • TotalSeq-B Mouse Myeloid Cocktail, V1.0, (BioLegend, Cat. No. 199904)
  • TotalSeq-B Hashtags
  • TotalSeq-B Nuclear Hashtags
  • TotalSeq-C Human Universal Cocktail, V2.0, (BioLegend, Cat No. 399910)
  • TotalSeq-C Human Universal Cocktail, V1.0, (BioLegend, Cat No. 399905)
  • TotalSeq-C Human Essential Cocktail, V1.0, (BioLegend, Cat No. 399914)
  • TotalSeq-C Human TBNK Cocktail, (BioLegend, Cat. No. 399903)
  • TotalSeq-C Mouse Universal Cocktail, V1.0, (BioLegend, Cat. No. 199903)
  • TotalSeq-C Hashtags

 

Best Practices and Important Considerations for Best Results

 

Surface Staining

 

Cell Viability

  • High viability prior to staining is critical for optimal performance. Ideal cell viability is >95%. Low cell viability is associated with poor single cell sequencing data. If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation. For samples with low viability, we recommend using BioLegend’s MojoSort™ Human or Mouse Dead Cell Removal Kits to enrich for viable cells prior to staining. Contact BioLegend Technical Services with any questions regarding cell viability.

 

Cell washing

  • When washing cells, it is extremely important to thoroughly decant the wash buffer, and upon the addition of new wash buffer, that the cell pellet is resuspended either with pipette mixing or gentle vortexing. When decanting, pour off the wash buffer in a single firm, but not forceful motion. Following decanting, continue to hold the tube inverted and remove remaining droplets on the lip of the tube by gently dabbing with clean paper towel before returning the tubes to an upright position. This technique should be used during all cell washes.

 

Optimal cell staining with TotalSeq™ antibodies

  • Our liquid, single-antibody TotalSeq conjugates require optimization of staining concentration (titration) to obtain best results, as performed by the end user of the antibodies. This includes TotalSeq antibodies targeting intracellular proteins of interest. Optimization of antibody staining concentration is essential to obtain good quality data in antibody-based applications such as surface and intracellular staining using TotalSeq antibodies.
  • Antibody titration for sequencing-based applications using TotalSeq antibodies is best performed using matching procedures as much as possible. This means titration by sequencing if possible. In the absence of titration-by-sequencing, it may be possible to replicate this protocol using fluorescent antibodies of the same clone and titrate via flow cytometry. However, this may prove challenging for TotalSeq intracellular antibodies as optimal concentration via flow cytometry may not correlate well with optimal concentration for detection of intracellular targets by sequencing. In general, we have found that most intracellular antibodies can be used at a concentration between 0.1 – 0.5 µg. BioLegend can provide recommended concentration ranges for most TotalSeq antibodies, including intracellular antibodies, and these can be obtained by contacting BioLegend Technical Services.
  • Throughout this protocol the term “antibody pool” is defined as a user-created antibody pool of titrated single TotalSeq antibodies.
  • BioLegend’s lyophilized TotalSeq-B and C antibody cocktails have been validated for use with this application. All cocktails have been validated to stain 2.5 x 105 - 2 x 106 cells. The cocktails are optimized for specific staining volumes. TotalSeq Human TBNK Cocktails are optimized to stain in a 100 µL total staining volume, and all other TotalSeq Cocktails have been optimized to stain in a 50 µL total staining volume. It is important that final staining volume, regardless of cell number used, does not exceed 100 µL for the TBNK cocktails and 50 µL for all other TotalSeq-B and -C lyophilized cocktails. Sample specific variations on cocktail performance are expected with smaller or larger cell input numbers and customers should verify adequate sensitivity in cocktail performance in their experiment.

    Critical:

    Although TotalSeq Cocktails have been validated to stain as few as 2.5 x 105 cells, it is critical for this protocol to start surface antibody staining with a minimum of 2 x 106 cells. If you do not have enough cells to achieve this, it is strongly recommended that you hashtag and pool samples to start surface staining with 2 x 106 cells.

 

Intracellular staining

  • Working solutions used for the nuclear staining section must be prepared fresh on the day of use and kept at room temperature. Once prepared, incubate working solutions at room temperature for 15 minutes before use. Prepare only the amount needed (with 10% excess) and discard any leftover solution.
  • The TotalSeq Nuclear Perm/Wash Component A may precipitate during storage, but this will not compromise performance.
  • The prepared TotalSeq Permeabilization and Washing Base (A+B) can be kept at 4°C for 1 month.
  • The TotalSeq Sample Resuspension Buffer should be stored and used at 4°C.
  • The addition of ribonuclease (RNase) inhibitors VRC and PVSA to prepared solutions used in this protocol is critical. The RNase inhibitors and quantities indicated below have been optimized for use in each respective buffer detailed in this protocol. Any alteration will require further customer validation. Samples known for having high RNase content might require more inhibitors than indicated.
  • Prior to VRC use, it is recommended to thaw the flask in a room temperature water bath for 30 minutes and vortex to resuspend any precipitated material. Once dissolved, use RNase-free consumables to make 0.5 mL aliquots and store at -20°C. Aliquots will require heating to 65°C and vortexing before use.
  • During nuclear staining, it is essential to use proper techniques to avoid RNase contamination. Perform all steps in an RNA-free designated area thoroughly cleaned with RNaseZap or RNase Away. Use only RNase-free consumables and always wear gloves.
  • The use of TotalSeq Fixation Buffer DOES NOT eliminate the need for the 10x Genomics fixation step detailed in the 10x Genomics CG000478 Demonstrated Protocol Cell & Nuclei Fixation Chromium Fixed RNA Profiling. The second fixation step is essential for Antibody Derived Tag (ADT) and RNA detection and should be performed following intracellular staining as detailed at the end of this protocol.

Protocol

 

Cell Surface Staining

 

  1. Prepare cell suspension with preferred or recommended method

    Notes:

    • This protocol has been optimized using fresh human PBMCs isolated using density gradient centrifugation. Whole blood or lysed whole blood is not recommended. If using cells isolated with a different procedure, users may need to verify the antibody staining pattern using alternative methods.
    • BioLegend has not tested this protocol using single-cell suspensions derived from enzymatically digested tissue. Enzymatic digestion may result in alterations of surface protein epitopes and impact staining with TotalSeq antibodies. Optimization of staining conditions and concentrations may be required.
    • Transcription factors are cell-, stage-, and activation-dependent. Based on your experimental set up, you may find abundant expression. Thus, it is always advisable to include a biological positive control. Cell lines are usually the ideal positive control. Refer to sources like “The Human Protein Atlas” to identify specific cell lines that are positive for your desired target. Previous validation by flow cytometry is recommended.
  2. Count and assess cell viability
    1. Using your preferred method, carefully count all cells and assess cell viability. It is critical to start cell surface protein staining with 2 x 106 cells.

      Notes:

      • The recommended starting cell number is required to ensure sufficient cells are available for the nuclear protein staining step. Using fewer cells can make it challenging to see the cell pellet after fixation and during wash centrifugation steps.
      • If you anticipate having fewer than 2 x 106 cells for a single sample at the start of this protocol, please reach out to BioLegend Technical Services for guidance on using hashtags to combine samples after cell surface staining.
      • Contact BioLegend Technical Services with any questions regarding cell viability. Ideal cell viability is >95%. Low cell viability is associated with poor single cell sequencing data. If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation. We recommend live cell enrichment be performed prior to cell surface protein staining.
  3. Dilute cells and Fc receptor blocking
    1. If using an antibody pool or the TotalSeq TBNK Cocktail:
      1. For human cells, dilute 2 x 106 cells in 45 μL of Cell Staining Buffer in a 12 x 75 mm flow cytometry tube and then add 5 μL of Human TruStain FcX™ for a total volume of 50 μL.
      2. For mouse cells, dilute 2 x 106 cells in 49.5 μL of Cell Staining Buffer in a 12 x 75 mm flow cytometry tube and then add 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) for a total volume of 50 μL.
    2. If using any other TotalSeq-B or -C Cocktail:
      1. For human cells, dilute 2 x 106 cells in 20 μL of Cell Staining Buffer in a 12 x 75mm flow cytometry tube and then add 5 μL of Human TruStain FcX™ Fc blocking reagent for a total volume of 25 μL.
      2. For mouse cells, dilute 2 x 106 cells in 24.5 μL of Cell Staining Buffer in a 12 x 75 mm flow cytometry tube and then add 0.5 μL of TruStain FcX™ PLUS (anti-mouse CD16/32) for a total volume of 25 μL.
    3. Incubate for 10 minutes at 4°C.
    4. While cells are incubating in Fc Block, proceed to step 4 - Stain cells with cell surface antibodies.

       

      Cocktail Type Cell Type Cell # Cell Staining Buffer Blocking Solution Total Staining Volume
      If using an Antibody pool or the TotalSeq TBNK Cocktail Human cells 2 x 106 45 μL Human TruStain FcX
      5 μL      		 50 μL
      Mouse cells 2 x 106 49.5 μL TruStain FcX PLUS
      0.5 μL      		 50 μL
      Any other TotalSeq B or C Cocktail Human cells 2 x 106 20 μL Human TruStain FcX
      5 μL      		 25 μL
      Mouse cells 2 x 106 24.5 μL TruStain FcX PLUS
      0.5 μL      		 25 μL
  4. Stain cells with cell surface antibodies

    Notes:

    • If you plan to multiplex samples using TotalSeq hashtags, there are two commonly used approaches to staining samples with hashtag antibodies.
      • Option 1: Individual samples are stained with TotalSeq antibodies (user-created antibody pool or with our pre-optimized TotalSeq-B or -C cocktails) and hashtags in a single step, washed three times to remove unbound antibodies, and subsequently pooled. See figure 1A.
      • Option 2: Involves first staining individual samples with hashtags, washing three times, sample pooling, and then staining with the TotalSeq antibodies (user-created antibody pool or with our pre-optimized TotalSeq-B or -C Cocktails), followed by another three washes. See figure 1B.
    • Important: If you do not have enough cells per sample to meet the recommended input of 2 × 106 cells for the cell surface antibody staining step, we recommend using Option 2. After completing the final wash of each individually hashed sample, you can pool the samples to reach a total of 2 × 106 cells for the surface staining step.
    • Whenever possible, we recommend option 1, as option 2 can result in increased sample loss and reduced cell viability due to multiple wash steps. If using option 2, we do not advise reducing sample washing as this may result in higher background and poor data quality. Additional information regarding use of TotalSeq hashtag antibodies can be found here - Efficient Multiplexing With TotalSeq Hashtags.
    • If cell hashing samples simultaneously with our TotalSeq Cocktails, or if you plan to “Spike-In” additional antibodies to the cocktails, please refer to our TotalSeq Antibody Cocktail “Spike-in” Guidance protocol prior to reconstitution of the lyophilized cocktail to determine how to make the Cell Staining Buffer and spike-in reconstitution mix.
    • For assistance in de-multiplexing after sequencing, please contact BioLegend Technical Services.

     

     

     

    A.

    B.

    Figure 1: A. Staining with TotalSeq antibodies and hashtags in a single step before pooling samples. B. Staining individual samples with hashtag reagents followed by pooling and staining with additional TotalSeq antibodies in subsequent steps.

     

    1. Staining individual samples with TotalSeq Hashtag Antibodies for sample pooling

      Notes:

      • If sequentially staining individual samples with hashtag antibodies, pooling samples, then staining with a TotalSeq antibody pool or one of our TotalSeq cocktails, follow the steps below before staining your samples with an antibody pool or cocktail (figure 1B). Please note that when sequentially staining, cell recovery may be diminished, and cell viability may be negatively impacted in some cases.
      • Proceed to step 4.2 if using an Antibody Pool, or to step 4.3 if using a TotalSeq-B or TotalSeq-C Cocktail, if any of the following conditions apply:
        • If you are not using TotalSeq Hashtags.
        • If you are staining cells simultaneously with an antibody pool or TotalSeq-B or -C cocktail and hashtags in a single step before pooling samples (figure 1A).
      1. Make a unique hashtag staining solution for each sample using a titrated amount of each hashtag in Cell Staining Buffer. Add the calculated amount of each hashtag antibody to a 1.5 mL low protein binding microcentrifuge tube and bring up the total volume with Cell Staining Buffer to 50 µL if using an antibody pool or TotalSeq TBNK Cocktail. If using any other TotalSeq-B or -C Cocktail, bring the total volume up to 25 µL.
      2. Centrifuge the hashtag solution at 14,000 x g at 2 – 8°C for 10 minutes before adding to the cells to ensure removal of protein aggregates.
      3. Carefully pipette out the prepared hashtag solution, avoiding the bottom of the tube, and add it to the 50 µL or 25 µL blocked cell suspension.
      4. Incubate for 30 minutes at 4°C.
      5. Wash cells by adding 3 mL of Cell Staining Buffer and mix by gently pipetting 5 times. Centrifuge at 4°C for 5 minutes at 400 – 600 x g depending on your sample type. Carefully decant the supernatant without disturbing the cell pellet.
      6. Repeat wash twice for a total of 3 washes.

        Notes: It is extremely important to thoroughly decant the wash buffer and resuspend the cell pellet either with pipetting or gentle vortexing. Discard supernatant with a single firm, but not forceful motion. Proceed to absorb any remaining liquid on the lip of the tube with a clean paper towel.

      7. After the final wash, resuspend each sample in 100 µL of Cell Staining Buffer and verify cell concentration for each sample.
      8. Based on each sample’s cell concentration, combine an equal number of cells from each sample to achieve 2 x 106 cells total in a 1.5 mL low protein binding microcentrifuge tube. The final volume will be dependent on the specific staining reaction size of the antibody pool or cocktail being used.
        • If staining cells with an antibody pool or a TotalSeq TBNK cocktail and the volume of combined cells is less than 50 µL, adjust volume with Cell Staining Buffer up to 50 µL to achieve 2 x 106 cells/50 µL. If the final volume of combined cells is greater than 50 µL, centrifuge at 4°C for 5 minutes at 400 – 600 x g. Following centrifugation, adjust volume to 50 µL by removing Cell Staining Buffer. After addition or removal of Cell Staining Buffer, gently resuspend cells by pipetting.
        • If staining cells with any other TotalSeq-B or -C Cocktail and the volume of combined cells is less than 25 µL, adjust volume with Cell Staining Buffer up to 25 µL to achieve 2 x 106 cells/25 µL. If the final volume of combined cells is greater than 25 µL, centrifuge at 4°C for 5 minutes at 400 – 600 x g. Following centrifugation, adjust volume to 25 µL by removing Cell Staining Buffer. After addition or removal of Cell Staining Buffer, gently resuspend cells by pipetting.

        Examples:

        • If you combined 6 samples at 10 µL each for a total of 60 µL and used an antibody pool or TotalSeq TBNK Cocktail, you would remove 10 µL after centrifugation for a total volume of 50 µL, resulting in 2 x 106 cells/50 µL.
        • If you combined 4 samples at 10 µL each for a total of 40 µL and used a TotalSeq Universal Cocktail to stain cells, you would remove 15 µL of buffer after centrifugation for a total of 25 µL, resulting in 2 x 106 cells/25 µL.
      9. Proceed with staining your combined samples using either section 4.2 (Cell staining with TotalSeq Antibody Pool and TotalSeq Hashtags), or section 4.3 (Cell staining with TotalSeq-B or -C Cocktail and TotalSeq Hashtags) below.

        Cells will already be stained with TotalSeq Hashtag Antibodies; do not add additional Hashtag Antibodies to the TotalSeq-B or -C antigen specific antibody pool or cocktail.
    2. Cell staining with TotalSeq Antibody Pool and TotalSeq Hashtags
      1. Prepare antibody pool by combining titrated amounts of each specific TotalSeq antibody in a 1.5 mL low protein binding microcentrifuge tube. For more information regarding titration of TotalSeq antibodies, please read Tips and Tricks for Titrating TotalSeq Antibodies. If you have additional questions, please reach out to BioLegend Technical Services.
      2. If cell hashing samples simultaneously with a TotalSeq Antibody Pool, we recommend adding a titrated amount of cell hashing antibody into each respective sample’s TotalSeq antibody pool (figure 1A). If you are not hashing, proceed to the next step
      3. If the antibody pool volume is less than 50 µL, adjust volume with Cell Staining Buffer up to 50 µL. If the volume of the pool is above 50 µL, no volume adjustment is necessary.
      4. Centrifuge the antibody pool at 14,000 x g at 2 – 8°C for 10 minutes before adding to the cells. This is critical to ensure removal of protein aggregates.
      5. Carefully pipette 50 µL of the prepared antibody pool, avoiding the bottom of the tube, and add the TotalSeq antibody pool to the 50 µL blocked cell suspension from section 3 OR the 50 µL hashed cell suspension from section 4.1.9.
      6. Incubate for 30 minutes at 4°C.
      7. Following incubation proceed to step 5, Wash Cells. Surface and Hashtag antibody-stained samples can be pooled after washing.
    3. Cell staining with TotalSeq-B or -C Cocktail and TotalSeq Hashtags
      • If staining cells with a TotalSeq TBNK or any other TotalSeq-B or -C cocktail, follow the reconstitution steps below.
      1. Equilibrate the lyophilized panel vial(s) to room temperature for 5 minutes.
      2. Place lyophilized panel vial in an empty 1.5 mL low protein binding microcentrifuge tube, and spin down at 10,000 x g for 30 seconds at room temperature.
      3. If cell hashing samples simultaneously with a TotalSeq Cocktail, please refer to the “Spike-In” Guidance protocol prior to reconstitution of the lyophilized cocktail to determine how to make the Cell Staining Buffer and spike-in reconstitution mix. Then proceed to the next step, 4.3.4, to reconstitute the lyophilized cocktail. If not hashing, or if your samples are already stained with hashtags, proceed to the next step, 4.3.4.
      4. If using the TBNK cocktail, rehydrate by adding 50 µL of Cell Staining Buffer, or the Cell Staining Buffer plus hashtag mix. If using any other TotalSeq-A Cocktail, rehydrate by adding 27.5 µL of Cell Staining Buffer or the Cell Staining Buffer plus hashtag mix. Replace the cap and vortex for 10 seconds.
      5. Incubate at room temperature for 5 minutes.
      6. Vortex again and spin down at 10,000 x g for 30 seconds at room temperature.
      7. Transfer the entire volume (50 µL for the TotalSeq-A TBNK Cocktail or 27.5 µL for any other TotalSeq-B or C Cocktail) to a 1.5 mL low protein binding microcentrifuge tube.
      8. Centrifuge at 14,000 x g for 10 min at 4°C. Important: Centrifugation of the reconstituted cocktail at 14,000 x g at 2 – 8°C for 10 minutes is critical to ensure removal of antibody aggregates.
      9. Transfer 50 µL or 25 µL of the reconstituted cocktail to the tube containing 50 µL or 25 µL of Fc Receptor-blocked cells or hashed cells, taking care to avoid the bottom of the tube. The final staining volume will be 100 µL for cells stained with the TotalSeq TBNK Cocktail, or 50 µL for cells stained with any other TotalSeq-B or C Cocktail. Mix by gently pipetting 5 times.
      10. Incubate for 30 minutes at 4°C.
      11. Following incubation proceed to step 5, Wash Cells. Surface and Hashtag antibody stained samples can be pooled after washing.
  5. Wash cells
    1. Add 3 mL of Cell Staining Buffer and mix by gently pipetting 5 times. Centrifuge at 4°C for 5 minutes at 600 x g. Carefully decant the supernatant without disturbing the cell pellet. Repeat step for a total of 2 washes.

      Note: It is important to thoroughly decant the wash buffer and resuspend the cell pellet either with pipetting or gentle vortexing. Discard supernatant with a single firm, but not overly forceful motion. Proceed to absorb any remaining liquid on the lip of the tube with a clean paper towel.

    2. Carefully decant the supernatant without disturbing the cell pellet and gently mix the cells by pulse vortexing the cell pellet at medium intensity in the residual volume.
    3. Add 3 mL of TotalSeq Cell Resuspension Buffer and mix by gently pipetting 5 times. Centrifuge at 4°C for 5 minutes at 600 x g.
    4. Carefully decant the supernatant without disturbing the cell pellet and gently mix the cells by pulse vortexing the cell pellet at medium intensity in the residual volume. Transfer the cells to a 1.5 mL low protein binding microcentrifuge tube.
    5. Carefully count all cells and assess viability with your preferred method using a 1:5 dilution in a new 1.5 mL low protein binding microcentrifuge tube.
    6. If you did not hashtag your samples or you hashtagged your samples and pooled prior to cell surface antibody staining proceed to step 5.8.
    7. If you simultaneously hashtagged and stained your samples with and antibody pool or TotalSeq Cocktail, combine an equal number of cells from each sample to achieve 2 x 106 cells total. Bring the total volume of the combined samples up to 1 mL with cold TotalSeq Cell Resuspension buffer.
    8. Centrifuge tubes at 600 x g for 10 minutes at 4°C.
    9. Carefully remove all the supernatant using a P200 pipette without disturbing the cell pellet.
    10. Resuspend the cell pellet in 100 μL of cold TotalSeq Cell Resuspension Buffer. Keep samples on ice until needed for intracellular staining.
  6. Solution and intracellular antibody pool preparation

    Critical:

    • If you plan to complete the nuclear staining portion of this protocol in one day prepare all solutions below before initiating step 7, Fixation.
    • If you would like to break the protocol up into 2 days, you can stop after fixation, and, in this case, only prepare the TotalSeq Diluent Solution and Sample Storage Buffer. Prepare remaining solutions the following day before starting the protocol.
    • Start by preparing all working solutions without VRC. VRC should be added warm to each solution as the final component.
    • Incubate solutions for 15 minutes at room temperature before using.
    1. TotalSeq Diluent Solution

       

      Reagent Stock concentration Final Concentration Volume (1 reaction + 10%)
      TotalSeq Diluent 1X 1X 825 μL
      VRC 200 mM 2 mM 11 μL
      Total     836 µL

       

      *If you are stopping and performing the nuclear staining on day 2, only prepare the TotalSeq Diluent and Sample Storage Buffer (preparation described in step 8, Overnight Sample Storage).

    2. Totalseq Permeabilization and Washing Base (A+B)

       

      Reagent Stock concentration Final Concentration Volume (1 reaction + 10%)
      TotalSeq Perm Component A NA NA 1.4 mL
      TotalSeq Perm Component B NA NA 2.8 mL
      Total     4.2 mL

       

      *Prepare the day of nuclear staining.

    3. 1X TotalSeq Nuclear Permeabilization and Washing Buffer

       

      Reagent Stock concentration Final Concentration Volume (1 reaction + 10%)
      Totalseq Permeabilization and Washing Base (A+B) 3.33X 1X 3.9 mL
      VRC 200 mM 2 mM 130 μL
      PVSA 30% 0.125% 44 μL
      Water - - 8.9 mL
      Total     12.974 mL

       

      *Prepare the day of nuclear staining.

    4. Blocking Solution

       

      Reagent Stock concentration Final Concentration Volume (1 reaction + 10%)
      Totalseq Permeabilization and Washing Base (A+B) 3.33X 1X 33 μL
      VRC 200 mM 3 mM 1.65 μL
      PVSA 30% 0.15% 0.55 μL
      TruStain FcX Block NA NA 5.5 μL
      True-Stain Monocyte Blocker NA NA 5.5 μL
      UltraPure Salmon Sperm DNA 10 mg/mL 0.75 mg/mL 8.25 μL
      Water - - 55.6 μL
      Total     110.1 µL

       

      *Prepare the day of nuclear staining.

    5. Intracellular Staining Solution

      TotalSeq-B or -C Intracellular Antibody Pool Preparation (Prepare day of nuclear staining)
      1. In a new, clean, 1.5 mL low protein binding microcentrifuge tube, prepare the intracellular antibody pool using titrated amounts of each TotalSeq intracellular antibody for each sample. We have found that most TotalSeq intracellular antibodies can be used at a concentration between 0.1 – 0.5 μg. For more information regarding TotalSeq antibody concentrations, please reach out to BioLegend Technical Services.
      2. After antibodies have been combined, bring the volume up to 37 µL with molecular grade PBS. DO NOT dilute the antibodies with 1X TotalSeq Nuclear Permeabilization and Washing Buffer in this step; PBS must be used. Dilution with 1X TotalSeq Nuclear Permeabilization and Washing Buffer during this step can contribute to increased aggregate formation.
      3. Centrifuge the antibody pool at 21,000 x g at 2 – 8°C for 10 minutes. This is critical to ensure removal of protein aggregates.
      4. Following centrifugation, carefully pipette out 35 μL of the prepared antibody pool, avoiding the bottom of the tube, and add the TotalSeq antibody pool to a new, clean, low protein binding microcentrifuge tube.
      5. Proceed with adding Totalseq Permeabilization and Washing Base (A+B) as described in the table below to the antibody pool and mix by pipetting 5 times.

         

        Reagent Stock concentration Final Concentration Volume (1 reaction + 10%)
        Totalseq Permeabilization and Washing Base (A+B) 3.33X 1X 15 μL
        Antibody pool 1X 1X 35 μL

         

        *Prepare the day of nuclear staining. Must be used the day of staining; do not store overnight.

      6. If antibody pool volume is larger than 37 µL, increase the volume of Totalseq Permeabilization and Washing Base (A+B). Use the following formula to determine the volume of Totalseq Permeabilization and Washing Base (A+B) to add to the antibody pool.

        Volume of Permeabilization and Washing Base (A+B) = 15 * antibody pool volume/35
         
      7. The Antibody Pool can be stored at room temperature until ready for use in step 10, Blocking, staining, and washing below. Stain cells with intracellular antibodies. The Antibody Pool should be made and used on the same day; do not store overnight.
  7. Fixation
    1. Transfer the cell suspension to a 12 x 75 mm flow cytometry tube. Pulse vortex the cell suspension 5 times, 1 second pulse each.
    2. Add 200 μL of the TotalSeq Nuclear Fixation Buffer. Pulse vortex the cell suspension 5 times, 1 second pulse each. Incubate for 5 minutes at room temperature.

      Note: Following this step cells should not be vortexed, cells should only be mixed or resuspended by gently pipetting.

    3. Add 760 μL of the prepared Diluent Solution. Gently mix 5 times using a P1000 pipette set to 950 μL. Incubate cells at room temperature for 1 hour.
    4. Proceed to step 9, Nuclear permeabilization and washing. Optionally the protocol can be stopped at this step. If you stop now, follow the optional overnight sample storage step 8, Overnight sample storage.
  8. (Optional) Overnight sample storage
    1. Prepare the Sample Storage Buffer.

       

      Sample storage buffer

       

      Reagent Stock concentration Final Concentration Volume (1 reaction)
      Cyto-Last Buffer - - 1035 μL
      VRC 200 mM 5 mM 27.5 μL
      PVSA 30% 1% 36.7 μL

       

      *Only prepare if storing samples overnight.

    2. Centrifuge sample tubes at 1000 x g for 5 minutes at room temperature.
    3. Carefully decant the supernatant without disturbing the cell pellet.
    4. Resuspend fixed cells in 1 mL of prepared Sample Storage Buffer by gently pipetting 5 times with a P1000 pipette set to 450 µL.
    5. Store samples overnight at 4°C. Following overnight storage the VRC may precipitate, this will not impact subsequent steps or results.
    6. On the following day, prepare the 1X TotalSeq Nuclear Permeabilization and Washing Buffer, Blocking Solution and Intracellular Staining Solution.
    7. Centrifuge the sample tubes at 1000 x g for 5 minutes at room temperature. Carefully decant the supernatant without disturbing the cell pellet. Resuspend the cells in residual volume by gently pipetting 5 times with a P200 pipette. Proceed to step 9.
  9. Nuclear permeabilization and washing
    1. Add 2 mL of the prepared 1X TotalSeq Nuclear Permeabilization and Washing Buffer. Cap the tubes securely and mix by inverting 10 times.

      Note:

      If you did not store your samples overnight, the 2 mL of 1X TotalSeq Nuclear Permeabilization and Washing Buffer will be added to the cells in fixation solution.

    2. Centrifuge the sample tubes at 1000 x g for 5 minutes at room temperature.
    3. Carefully decant the supernatant without disturbing the cell pellet.
    4. Add 1000 μL of the 1X TotalSeq Nuclear Permeabilization and Washing Buffer. Resuspend the cells by gently pipetting 5 times with a P1000 pipette set at 950 µL. Transfer the resuspended cells to a labeled 1.5 mL low protein binding microcentrifuge tube.
    5. Centrifuge the 1.5 mL low protein binding microcentrifuge tube at 1000 x g for 5 min at room temperature. Make note of the expected cell pellet location based on the angle of the tube in the centrifuge as the cell pellet may be difficult to see after centrifugation.
    6. Carefully remove all the supernatant using a pipette without disturbing the cell pellet. The cell pellet may be difficult to see, so extra care should be taken. Based on your earlier assessment of expected pellet location, pipette from the opposite side. We suggest removing the supernatant in increments rather than trying to remove all at once.
  10. Blocking, staining, and washing
    1. Add 100 μL of the prepared Blocking Solution. Gently pipette mix 15 times using a P200 pipette set to 90 μL. Incubate at room temperature for 15 minutes.
    2. Add 50 μL of the Intracellular Antibody Staining Solution. Gently pipette mix 15 times using a P200 set to 140 μL. Incubate at room temperature for 1 hour.
    3. Add 1 mL of the prepared 1X TotalSeq Nuclear Permeabilization and Washing Buffer. Gently pipette mix the cell suspension 5 times using a P1000 pipette set to 500 µL. Transfer all the volume to a new 12 x 75mm flow cytometry tube.
    4. Add 2 mL of the prepared 1X TotalSeq Nuclear Permeabilization and Washing Buffer. Cap tubes securely and mix by inversion 10 times.
    5. Centrifuge tubes at 1000 x g for 5 minutes at room temperature. Carefully decant the supernatant without disturbing the cell pellet. Resuspend the cells in residual volume with a P200 carefully pipetting 5 times.
    6. Repeat steps 10.3 - 10.5 two times for a total of 3 washes.
    7. Add 500 μL of the prepared 1X TotalSeq Permeabilization and Washing Buffer. Resuspend cell pellet by gently pipetting 15 times.
    8. Transfer resuspended cells to a 1.5 mL low protein binding microcentrifuge tube.
    9. Centrifuge the 1.5 mL low protein binding microcentrifuge tube at 1000 x g for 5 min at room temperature.
    10. Carefully remove all the supernatant using a pipette without disturbing the cell pellet.
    11. Proceed immediately to step C in the Sample Fixation section of the 10x CG000478 Demonstrated Protocol Cell & Nuclei Fixation Chromium Fixed RNA Profiling.

       

      Library Type Minimum Sequencing Depth (reads/cell))
      Cell Surface and Intracellular Protein Library 10,000

       

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